1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the selection of reverse transcription primer pools to avoid amplification of a small group of unwanted transcripts, while reverse transcribing most other transcripts in a sample.
2. Description of Related Art
With the advent of novel high-throughput approaches, such as microarrays, researchers can now measure changes of the expression profiles of thousands of genes in a single experiment. As the first step, RNAs of interest are usually converted to cDNA. This conversion is performed with a reverse transcription (RT) reaction. Currently two types of oligonucleotides primers, oligo dT and random hexamers are commonly used to anneal to RNA molecules to start the RT reactions. Unfortunately, both priming methods have major limitations.
Random primers are commonly used in RT reactions for total RNA. Typically, most of the RNA in a sample is rRNA, whereas other transcripts (e.g. mRNAs) comprise only a small percentage of total RNA. As a result, the overwhelming majority of final cDNA products are from rRNAs. The presence of these rRNA-derived products may be detrimental to many downstream applications. For example, background signals increase significantly in microarray hybridizations in the presence of cDNA products from rRNAs. Removing rRNA prior to microarray hybridization results in a higher percentage of present calls, which is an indication of better array sensitivity. rRNA removal also results in increased sample correlations/concordance among array replicates. The benefit of rRNA removal is even more obvious for amplified RNA samples.
Various methods have been applied for rRNA removal. For example, Affymetrix Inc. introduced a procedure for removing rRNA by enzymatic digestions. However, the whole procedure is both time consuming and expensive to researchers. Alternatively, mRNA may be enriched by removing rRNA molecules with magnetic beads. rRNA specific oligonucleotide probes are attached to magnetic beads, which are incubated with total RNA. In this way, rRNA is captured by the beads and later removed by centrifugation. Ambion provides a kit for the removal of bacterial RNA (MICROBExpress); the Ribo-Minus kit is available from Invitrogen for human and mouse rRNA removal. Thus, while it is possible to deplete rRNA from a sample, the extra step of rRNA removal can complicate experiments and introduce additional cost.
In contrast to the random priming strategy, oligo-d(T) priming is widely used to enrich the mRNA population directly. Oligo dT primers anneal specifically to the poly(A) tail of mRNA molecules, and thus reverse transcription of rRNA is minimized. However, oligo d(T) primers are not suitable for all applications. For example, oligo d(T) are not suitable as RT primers for bacterial mRNA because most of them do not have poly(A) tails. In addition, many other interesting non-coding RNAs in the transcriptome, such as microRNAs (miRNAs) and siRNAs will not be covered. Partially degraded RNAs also cannot be fully transcribed using oligo d(T) primers. Degraded RNA is commonly encountered with most clinical human samples such as RNA collected from Formalin Fixed Paraffin Embedded (FFPE) samples or from tissues rich in nucleases.
In addition, the oligo-d(T) priming strategy introduces 3′ bias in cDNA synthesis because it is difficult to produce full-length cDNAs due to the limited RT extension capability. This is an especially serious problem for RT-based linear RNA amplification since only about 1 kilobase of 3′ sequences can be effectively amplified. In view of this problem, most microarray platforms are designed for the 3′ regions of the transcripts. For example, many Affymetrix probes are picked from the last 600 bases of the mRNA sequences. Unfortunately, this size limitation is a major drawback because researchers are unable to examine relevant biological information, such as alternative splicing, from the entire transcriptome. Affymetrix has launched a new GeneChip platform—whole genome tiling arrays, which are designed for profiling of the entire transcriptome. However, most existing RNA amplification products for GeneChips use oligo-d(T) as the RT primer and thus will not be suitable for the new full-transcript coverage arrays.